Epithelial cells assemble into mucosal sheets and act as the barrier between the external and internal milieu. To maintain this functional barrier, intestinal epithelial cells must establish segregated apical and basolateral domains. Alterations in these processes through losses in polarity can lead to neoplasia, while loss of proper microvillar assembly or maintenance can lead to malabsorbtion of nutrients and diarrheal disease. Our recent studies have demonstrated that vesicle trafficking proteins critically regulate the processes required for establishment and maintenance of apical polarity. Rab11-Family Interacting Proteins (Rab11-FIPs) mediate both directional trafficking and the establishment of apical polarity. The establishment of apical polarity involves proper phosphorylation of Rab11-FIP2 by the polarity-associated kinase MARK2/Par1b. We have recently demonstrated that Rab11-FIP1B/C is also a substrate for MARK2. While previous investigations have noted the importance of MARK2 phosphorylation in epithelial polarity, no studies have examined how MARK2- dependent phosphorylation events could influence intestinal epithelial cell polarity. In intestinal cells, we also have demonstrated that loss of Rab25, an epithelial-specific small GTP binding protein, in Rab25 KO mice promotes intestinal and colonic neoplasia and knockdown of Rab25 expression in CaCo-2 cells promotes a loss in polarized function and assumption of a more invasive phenotype. Rab25 expression is decreased in human colon cancers regardless of stage, suggesting a role for Rab25 loss in the early stages of carcinogenesis. In both the Rab25 KO mice and CaCo-2 cells, loss of Rab25 is associated with mis-trafficking of 1-integrin. In CaCo-2 cells, knockdown of Rab25 expression induces deficits in polarized function with both mistrafficking of integrins as well as decreases in 5-integrin transcription. We have hypothesized that components of plasma membrane recycling systems, Rab25 and Rab11-FIP1B/C and Rab11-FIP2, are critical mediators of both the establishment and maintenance of polarity and that defects in these pathways predispose to early carcinogenesis. To investigate our hypothesis we will pursue three specific aims: First, we will determine how Rab25 regulates intestinal polarity through regulation of ETV4-dependent gene transcription. Second, we will identify the roles of Rab11-FIP proteins in regulating polarity in intestinal cells. These studies will utilize novel cell culture models and phosphorylation site-specific antibodies. Third, we will determine the effects of the loss of Rab11-FIP2 and Rab11-FIP1B/C on intestinal cell polarity and differentiation in mice and intestinal enteroids in culture. These studies will utilize novel mouse models for floxed alleles for both Rab11-FIP1B/C and Rab11-FIP2. These investigations will establish how disruption of vesicle trafficking processes that regulate the establishment and maintenance of intestinal epithelial cell polarity may lead to carcinogenesis. !